Human neutrophils store a battery of hydrolytic enzymes that are primarily utilized for the degradation of foreign substances during the phagocytosis process, one of the main host defense mechanisms against infectious agents. Human neutrophil elastase (hereinafter referred to as elastase) is a major protein stored in the azurophilic granules of human polymorphonuclear granulocytes (Dewald et al. (1975) J. Exp. Med. 141: 709) and secreted upon inflammatory stimuli (Bonney et al. (1989) J. Cell. Biochem. 39:47). Elastase is a single-chain glycoprotein 218 amino acids in length (Sinha et al. (1987) Proc. Natl. Acad. Sci. USA 84:2228). Elastase has two N-glycosylation sites at positions Asn-95 and Asn-144. Its molecular weight is about 29,500 daltons, and its isoelectric point (pI) lies between 8-9. The crystal structure of elastase complexed with an inhibitor has been determined (Navia et al. (1989) Proc. Natl. Acad. Sci. USA 86:7).
Elastase is a serine protease with broad substrate specificity and, therefore, is a voracious enzyme able to digest many macromolecules found in connective tissues. For example, elastase can hydrolyze macromolecules such as elastin, type III and type IV collagen, and fibronectin. In addition to connective tissue components, many plasma proteins such as immunoglobulins, clotting factors and complement proteins can also be hydrolyzed by elastase.
Neutrophil turnover and phagocytosis result in the leakage of enzymes into the extracellular matrix where they can cause damage to connective tissues. Natural inhibitors of elastase are made in the body to control the damaging process.
Excess elastase activity has been implicated in various disease states, such as pulmonary emphysema (Kaplan et al. (1973) J. Lab. Clin. Med. 82:349; Sanders and Moore (1983) Am. Rev. Respir. Dis. 127:554), cystic fibrosis, rheumatoid arthritis (Barrett (1978) Agents and Actions 8:11), chronic bronchitis, bronchopulmonary dysplasia in premature infants, and adult respiratory distress syndrome (ARDS) (Weiland et al. (1986) Am. Rev. Respir. Dis. 133:218). In most cases, the pathogenesis of these diseases has been correlated with the inactivation or the insufficiency of natural inhibitors of elastase, whose primary role is to keep excess enzyme activity under control. Several natural proteinase inhibitors have been shown to be effective in regulating the activity of elastase. Human .alpha.-1-proteinase inhibitor (.alpha.-1-PI) is the primary protease inhibitor found in plasma (Heimburger et al. (1970) in Proceedings of the First International Research Conference on Proteinase Inhibitors, Walter de Gruyter, New York, pp 1-21). In some individuals, .alpha.-1-PI is present in unusually low amounts due to an underlying genetic defect; this low level causes familial emphysema (Garver et al. (1986) N. Engl. J. Med. 314:762). In other cases, the inhibitor is nonfunctional due to oxidative destruction by cigarette smoke (Janus et al. (1985) the Lancet i:152). Secretory leucocyte proteinase inhibitor (SLPI) is another elastase inhibitor found in all mucous secretions (Thompson & Ohlsson (1986) Proc. Natl. Acad. Sci. USA 83:6692) which is believed to be the major elastase inhibitor present in the upper airways of the lung. Recently, another natural elastase inhibitor, elafin, was characterized from human skin (Wiedow et al. (1990) J. Biol. Chem. 265:14791).
The development of elastase-specific inhibitors has been a major goal in the pharmaceutical industry for some time. As a result, different types of inhibitors have been developed. These include irreversible inhibitors such as peptide chloromethyl ketones (Powers et al. (1977) Biochim. Biophys. Acta 485:156), reversible inhibitors such as peptide boronic acids (Stone et al. (1990) Am. Rev. Respir. Dis. 141:47), cephalosporins (Doherty et al. (1986) Nature 322:192), and peptide aldehydes (Kennedy et al. (1987) Eur. J. Respir. Dis. 71:472). However, many of these inhibitors are nonspecific inhibitors of other serine proteases as well (Hemmi et al. (1985) Biochemistry 24:1841). Moreover, some of the more specific peptide-based inhibitors of elastase that have been developed suffer from a lack of oral availability (Williams et al. (1991) Am rev. Respir. Dis. 144:875). Some of the .beta.-lactam inhibitors have both stability problems and lack oral availability (Knight et al. (1992) Biochemistry 31:4980). In addition to synthetic inhibitors, biosynthetically derived naturally occurring inhibitors such as .alpha.-1-proteinase (Gadek et al. (1981) J. Clin. Invest. 68:1158), eglin C (Snider et al. (1985) Am. Rev. Respir. Dis. 132:1155), and SLPI (Gauthier et al. (1982) Biochim. Biophys. Acta 700:178; Gast et al. (1990) Am. Rev. Respir. Dis. 141: 889) are under development.
Polynucleotides including synthetic RNA homopolymers (Simon et al. (1988) Exp. Lung Res. 14:85), tRNAs and DNAs (Lestienne & Bieth (1983) Biochimie 65:49) have been shown to inhibit elastase to some extent. The enzyme inhibition caused by these polyanions is not likely due to simple electrostatic interactions, because other polyanions lacking hydrophobic constituents such as heparin and polyanionic polysaccharides have been shown to be ineffective inhibitors. Cell extracts of certain pneumococcal species (Pneumococci type I, type II smooth, and type II rough) yield high molecular weight RNAs upon autolysis that act as elastase inhibitors (Vered et al. (1988) Exp. Lung Res. 14:67).
A method for the in vitro evolution of nucleic acid molecules with high affinity binding to target molecules has been developed. U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled Systematic Evolution of Ligands by Exponential Enrichment, now abandoned, and U.S. Pat. No. 5,270,163, issued Dec. 14, 1993, and U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, both entitled Nucleic Acid Ligands (See also PCT/US91/04078) describe a fundamentally novel method for making a nucleic acid ligand for any desired target. Each of these applications, collectively referred to herein as the SELEX Patent Applications, is specifically incorporated herein by reference.
The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of the same general selection theme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting with a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield high affinity nucleic acid ligands to the target molecule.
The basic SELEX method may be modified to achieve specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, entitled Method for Selecting Nucleic Acids on the Basis of Structure, describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled Photoselection of Nucleic Acid Ligands, describes a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine, describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed "counter-SELEX". U.S. patent application Ser. No. 08/143,564, filed Oct. 25, 1993, entitled Systematic Evolution of Ligands by EXponential Enrichment: Solution SELEX, describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or delivery. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled High Affinity Nucleic Acid Ligands Containing Modified Nucleotides, which describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2'-positions of pyrimidines, as well as specific RNA ligands to thrombin containing 2'-amino modifications. U.S. patent application Ser. No. 08/134,028, supra, describes nucleic acid ligands to theophylline and caffeine containing one or more nucleotides modified with 2'-amino (2'-NH.sub.2), 2'-fluoro (2'-F), and/or 2'-Omethyl (2'-OMe).
The development of high affinity ligands capable of inhibiting elastase would be useful in the treatment of diseases such as pulmonary emphysema (Kaplan et al. (1973) supra; Sanders and Moore (1983) Supra), cystic fibrosis, rheumatoid arthritis (Barrett (1978) supra), chronic bronchitis, bronchopulmonary dysplasia in premature infants, and adult respiratory distress syndrome (ARDS) (Weiland et al. (1986) supra). Herein described are high affinity nucleic acid ligand inhibitors of elastase.